Autofluorescent Proteins Research Articles

Nanoscale imaging of major and minor ampullate silk from the orb-web spider Nephila Madagascariensis

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.

Design and Validation of Plasmid Vectors for Characterizing Protein–Protein Interactions in Spodoptera frugiperda Insect Cells

There are limited molecular biology resources for interrogating protein–protein interactions (PPIs) in insect cells. To address this deficiency, we developed plasmid vectors for localization, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays in Sf9 insect cells. Plasmids were designed to express a protein of interest as a fusion with epitope tags and autofluorescent proteins using the Gateway cloning system. Two robust interactors were utilized to validate this system: the nucleoprotein (N) and the phosphoprotein (P) of maize mosaic virus. The viral N was fused with the carboxy-terminal portion of eYFP and a FLAG epitope tag, and P was fused with the amino-terminal portion of eYFP and a myc epitope tag. The two expression plasmids were cotransfected into Sf9 cells, fluorescence microscopy was used to visualize BiFC, and co-IP was performed to confirm that this system was sensitive enough to detect PPI between the two proteins. BiFC was seen in cells cotransfected with N and P, and co-IP validated the interaction. This plasmid-based system can be used to investigate a variety of PPIs that occur in insects. We validated viral protein interactions that occur in the insect vector, which provides further insights into the biology of rhabdoviruses that are transmitted by insects. The ability to express viral and insect proteins in insect cells for studying PPIs with this streamlined system represents an advancement for protein research in insects. Future work will focus on identifying interacting viral and host proteins and discovery of targets for control of viruses and insect vectors. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Expression and function of the cdgD gene, encoding a CHASE\u2013PAS-DGC-EAL domain protein, in Azospirillum brasilense

The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Neutrophil fluorescence in clozapine users is attributable to a 14kDa secretable protein.

Clozapine is the only antipsychotic agent with demonstrated efficacy in refractory schizophrenia. However, use of clozapine is hampered by its adverse effects, including potentially fatal agranulocytosis. Recently, we showed an association between neutrophil autofluorescence and clozapine use. In this study, we evaluated the subcellular localization of clozapine‐associated fluorescence and tried to elucidate its source. Neutrophils of clozapine users were analyzed with fluorescence microscopy to determine the emission spectrum and localization of the fluorescence signal. Next, these neutrophils were stimulated with different degranulation agents to determine the localization of fluorescence. Lastly, isolated neutrophil lysates of clozapine users were separated by SDS‐PAGE and evaluated. Clozapine‐associated fluorescence ranged from 420 nm to 720 nm, peaking at 500‐550 nm. Fluorescence was localized in a large number of small loci, suggesting granular localization of the signal. Neutrophil degranulation induced by Cytochalasin B/fMLF reduced fluorescence, whereas platelet‐activating factor (PAF)/fMLF induced degranulation did not, indicating that the fluorescence originates from a secretable substance in azurophilic granules. SDS‐PAGE of isolated neutrophil lysates revealed a fluorescent 14kDa band, suggesting that neutrophil fluorescence is likely to be originated from a 14kDa protein/peptide fragment. We conclude that clozapine‐associated fluorescence in neutrophils is originating from a 14kDa soluble protein (fragment) present in azurophilic granules of neutrophils. This protein could be an autofluorescent protein already present in the cell and upregulated by clozapine, or a protein altered by clozapine to express fluorescence. Future studies should further explore the identity of this protein and its potential role in the pathophysiology of clozapine‐induced agranulocytosis.

Multifluorescence Live Analysis of Herpes Simplex Virus Type-1 Replication.

The possibility to label specific viral and cellular structures with live cell markers such as autofluorescent proteins has greatly contributed to our understanding of diverse steps of the virus life cycle, as it allows for monitoring virus replication in a spatial and temporal fashion. Here, we describe the multifluorescent live analysis of the multicompartment Herpes Simplex Virus Type-1 (HSV-1) by live cell confocal laser scanning microscopy.

Multivalent Chelators for In Vivo Protein Labeling

AbstractWith the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.

Multivalent Chelators for In Vivo Protein Labeling.

With the advent of single-molecule methods, chemoselective and site-specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme-based tags or autofluorescent proteins are short peptide-based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine-tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single-molecule approaches.

Stain-free LED scanning lifetime imaging system for diabetes modified tissue matrices.

In contrast to labor intensive and destructive histological techniques, intrinsic autofluorescence lifetimes of extra cellular matrix proteins can provide label-free imaging of tissue modifications in diseases, including the diabetic ulcers. However, decoupling the complex mixture of tissue fluorophores requires costly and complicated fluorescent lifetime instrumentation. Furthermore, a list of autofluorescent and fluorogenic proteins must be characterized to profile their changes during disease progression. Towards these goals, an imaging system based on frequency domain light-emitting diode (LED) modulation was designed and demonstrated, using off-the-shelf components in a low complexity design. The system was operated by coupling and imaging fluorescence intensities using a pair of objectives. The system's scanning and signal acquisition performances were optimized with respect to etendues. To study fluorescent proteins in diabetic ulcers, lifetimes from purified and pentosidine modified collagen I, collagen III, and elastin were measured. Pentosidine measurements showed a decrease in autofluorescent lifetimes while elevated collagen III in diabetic ulcers showed increased lifetimes. These lifetimes, plus future protein measurements enabled by our system, can serve as standards for developing a biophotonic model of diabetic ulcers. As a proof-of-concept, a 3 cm × 3 cm diabetic foot ulcer was imaged using the developed system. Phasor analysis was applied to aid the interpretation of lifetime images. As a result, a compact biophotonic imaging system targeting diabetic tissue was achieved, towards making the technique accessible for clinical histology.

Preparation, characterization and application of a protein hydrogel with rapid self-healing and unique autofluoresent multi-functionalities.

A smart hydrogel with dual self-healing and autofluoresent functionalities is presented. The protein hydrogel is fabricated by denaturing bovine serum albumin in a basic environment. Upon gelation, autofluorescence is induced and the protein hydrogel can be excited by a wide range of spectrum, ranging from 320 to 520 nm. It was also found that the as-prepared autofluorescent protein hydrogel possessed rapid and repetitive self-healing capability. Without any external stimulus, more than 90% recovery of the mechanical strength can be obtained within 10 min after destruction. Moreover, the as-prepared hydrogel exhibits excellent biocompatibility and cell attachment property after its pH adjustment to neutral pH, while both autofluorescence and self-healing properties were still retained. This study suggests a promising means to prepare multi-functional protein hydrogel with dual physicochemical functionalities, which holds great potential in biomedical related applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 81-91, 2019.

Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV)

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3′ genomic region sites, between ORF4′ and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2′, TRS4′ or TRS7. M013 insertion at the ORF4′/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2′ retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2′ at the ORF4′/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4′/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

Nucleocytoplasmic shuttling: The ins and outs of quantitative imaging.

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy

Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm−1 delocalized motion is observed, with dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.

Cellular localization and interactions of nucleorhabdovirus proteins are conserved between insect and plant cells

Maize mosaic virus (MMV), similar to other nucleorhabdoviruses, replicates in divergent hosts: plants and insects. To compare MMV protein localization and interactions, we visualized autofluorescent protein fusions in both cell types. Nucleoprotein (N) and glycoprotein (G) localized to the nucleus and cytoplasm, phosphoprotein (P) was only found in the nucleus, and 3 (movement) and matrix (M) were present in the cytoplasm. This localization pattern is consistent with the model of nucleorhabdoviral replication of N, P, L and viral RNA forming a complex in the nucleus and the subvirion associating with M and then G during budding into perinuclear space. The comparable localization patterns in both organisms indicates a similar replication cycle. Changes in localization when proteins were co-expressed suggested viral proteins interact thus altering organelle targeting. We documented a limited number of direct protein interactions indicating host factors play a role in the virus protein interactions during the infection cycle.

Hybrid Fluorescent Markers for Reporting and Biosensing on Demand

Deciphering the complex mechanisms controlling cells and organisms requires effective imaging systems and fluorescent probes to observe and quantify biomolecules in real time with high spatiotemporal resolution. A common strategy for imaging proteins is to fuse them to peptide or protein sequences that provide fluorescence, such as autofluorescent proteins. Recently, the fluorescence toolkit has been expanded with methods for labeling biomolecules with exogenously applied small synthetic fluorescent probes. These innovative technologies offer additional labeling refinement and broaden fluorescent labeling to more diverse cellular molecules. Selectivity is ensured through fusion to a genetic tag that binds selectively tailored fluorescent molecules. The modular nature of such an approach enables one to tune the synthetic part by molecular engineering, in order to address biological questions with the molecular diversity offered by modern chemistry. To be usable within living systems, the genetic tag must fold and function in various cellular compartments, while the fluorescent probes must ideally not show unspecific interaction/reaction with cell components. A way to avoid unspecific background in cells and achieve high imaging contrast is to use fluorescent probes that display no fluorescence until labeling occurs. Such probes are often called fluorogenic probes to highlight their ability to generate fluorescence upon reaction/interaction with their target. During this talk, I will present the development and applications of hybrid fluorescent markers that enable high imaging contrast relying on genetically encoded protein that bind and activate fluorogenic synthetic molecules (so-called fluorogens).

Protein Localization and Interaction Studies in Plants: Toward Defining Complete Proteomes by Visualization.

Protein interaction and localization studies in plants are a fundamental component of achieving mechanistic understanding of virus:plant interactions at the systems level. Many such studies are conducted using transient expression assays in leaves of Nicotiana benthamiana, the most widely used experimental plant host in virology, examined by laser-scanning confocal microscopy. This chapter provides a workflow for protein interaction and localization experiments, with particular attention to the many control and supporting assays that may also need to be performed. Basic principles of microscopy are introduced to aid researchers in the early stages of adding imaging techniques to their experimental repertoire. Three major types of imaging-based experiments are discussed in detail: (i) protein localization using autofluorescent proteins, (ii) colocalization studies, and (iii) bimolecular fluorescence complementation, with emphasis on judicious interpretation of the data obtained from these approaches. In addition to establishing a general framework for protein localization experiments in plants, the need for proteome-scale localization projects is discussed, with emphasis on nuclear-localized proteins.

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells.

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions.

Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.

Closed-form formulas vs. PDE based numerical solution for the FRAP data processing: Theoretical and practical comparison

Fluorescence recovery after photobleaching (FRAP) is a widely used method to analyze (usually using fluorescence microscopy) the mobility of either fluorescently tagged or autofluorescent (e.g., photosynthetic) proteins in living cells. The FRAP method resides in imaging the recovery of fluorescence intensity over time in a region of interest previously bleached by a high-intensity laser pulse. While the basic principles of FRAP are simple and the experimental setup is usually fixed, quantitative FRAP data analysis is not well developed. Different models and numerical procedures are used for the underlying model parameter estimation without knowledge of how robust the methods are, i.e., the parameter inference step is not currently well established.In this paper we rigorously formulate the inverse problem of model parameter estimation (including the sensitivity analysis), making possible the comparison of different FRAP parameter inference methods. Then, in a study on simulated data, we focus on how three different methods for inference influence the error in parameter estimation. We demonstrate both theoretically and empirically that our new method based on a solution of a general initial–boundary value problem for the Fick diffusion partial differential equation exhibits less bias and narrower confidence intervals of the estimated diffusion parameter, than two closed formula methods.